This invention relates to combining synergistic microorganisms to produce the signals that are necessary to facilitate germination and plant root colonization of mycorrhizae fungi. The colonization of the plant root by mycorrhizal fungi results in the increase of the availability of nutrients to plants, control of pathogens, and improved soil structure and/or soil quality. In particular, an illustrative embodiment of the invention relates to combining phytate as a nutrient source with a combination of Trichoderma virens, Bacillus amyloliquefaciens, and mycorrhizal fungi, including the following known species: Glomus intraradices, Glomus etunicatum, Glomus aggregatum, Glomus mosseae, for the purpose of replacing, duplicating, or enhancing the effect of standard phosphorus-containing fertilizer compounds, such as a 10-34-0, 9-18-9, 3-18-18 fertilizer or other NPK fertilizer combinations.
Phytic acid [known as inositol hexakisphosphate (IP6) or phytate when in salt form] is the principal storage form of phosphorus in many plant tissues, especially bran and seeds. Phytate is also a major form of organic phosphorus within the soil profile, typically constituting 20%-50% of soil organic phosphorus.
Trichoderma is a genus of fungi that contains about 20 species. Synonyms for the genus name include Aleurisma and Sporoderma. Trichoderma virens, which is also called Gliocladium virens, is a member of the genus. The natural habitats of these fungi include soil and plant material. A member of the genus, Trichoderma harzianum KRL-AG2 (ATCC 20847) also known as strain T-22, is used as a biocontrol agent that is applied as a seed or soil treatment or on cuttings and transplants. Strains of the species, Trichoderma virens, have also been used for control of damping off diseases in plants. For example, Trichoderma (Gliocladium) virens Gl-21 is known and commercially available at a reasonable price, and is being marketed under the trademark SoilGuard® 12G (EPA Registration Number: 70051-3 and EPA Establishment Number: 067250-IL-001). It is manufactured by Thermo Trilogy Corporation of Columbia, Md. Other known and commercially available Trichoderma virens strains include those having the following ATCC accession numbers: 10043, 10044, 10045, 13213, 13362, 204067, 204443, 204444, 204445, 20903, 20904, 20906, 24290, 42955, 44327, 44734, 48179, 52045, 52199, 58676, 58677, 58678, 62399, 64271, 74180, 9645, MYA-297, MYA-298, MYA-649 and MYA-650.
Bacillus is a genus of rod-shaped, gram-positive, aerobic or (under some conditions) anaerobic bacteria. Bacillus species are widely found in soil and water and some have been used to control plant diseases, including root rot. Bacillus amyloliquefaciens is a spore-forming member of the genus. Bacillus amyloliquefaciens L.L. Campbell strain F (ATCC 23350) is the type strain for the species. Other known and commercially available Bacillus amyloliquefaciens strains include those having the following ATCC accession numbers: 23842, 23843, 23844, 23845, 31592, 49763, 53495 and BAA-390 (Int. J. Sys. Bacteriol. 37:69-71, 1987; J. Bacteriol. 94:1124-1130, 1967).
In the past, before the name was officially changed to recognize that the microorganism was a new species, Bacillus amyloliquefaciens was also called Bacillus subtilis var. amyloliquefaciens by some investigators. A protease produced from Bacillus subtilis var. amyloliquefaciens is commonly used as a tenderizer for raw meat products. According to the U.S. Environmental Protection Agency (EPA), Bacillus subtilis var. amyloliquefaciens strain FZB24 is a naturally-occurring microorganism and widespread in the environment. Bacillus subtilis var. amyloliquefaciens FZB24 (EPA Registration Number: 72098-5 and EPA Establishment Number: 73386-DEU-001) is known and commercially available at a reasonable price, being marketed under the trademark Taegro® by Novozymes, Inc. of Brookfield, Wis.
An arbuscular mycorrhiza fungus is a type of mycorrhiza in which the fungus penetrates the cortical cells of the roots of a vascular plant. Arbuscular mycorrhizae fungi help plants to capture nutrients such as phosphorus, sulfur, nitrogen and micronutrients from the soil. It is believed that the development of the arbuscular mycorrhizal symbiosis played a crucial role in the initial colonization of land by plants and in the evolution of the vascular plants.
The development of arbuscular mycorrhizal fungi prior to root colonization, known as presymbiosis, comprises three stages: propagule germination, hyphal growth, and host recognition and appressorium formation. Propagule are thick-walled multi-nucleate resting structures. Arbuscular mycorrhizal fungi propagules may germinate given suitable conditions of the soil matrix, temperature, carbon dioxide concentration, pH, and phosphorus concentration. The germination of the propagule is not thought to be under direct control of the plant as propagules have been germinated under experimental conditions in the absence of plants both in vitro and in soil. However, the rate of propagule germination can be increased by plant host root exudates.
The growth of arbuscular mycorrhizal hyphae through the soil is controlled by host root exudates and the soil phosphorus concentration. Arbuscular mycorrhizal fungi colonization is higher in nutrient poor soils and decreased with the addition of phosphate fertilizer. Low soil phosphorus concentrations increase hyphal growth and branching as well as induce plant exudation of compounds which control hyphal branching intensity. Arbuscular mycorrhizal fungi also have chemotaxic abilities which enable hyphal growth toward the roots of a potential host plant.
A major challenge for the mycorrhizologist is to understand the extremely harmonious arbuscular mycorrhizal fungus host signaling mechanisms and the colonization process. This harmonious symbiotic relationship is reflected in the obligate biotrophic nature of the fungi, which cannot be cultured in the absence of a host. While success in achieving effective mycorrhizal associations with crop plants growing in sterilized soil has been achieved, the ultimate success for agricultural use of vesicular-arbuscular mycorrhizal (VAM) fungi will occur when they can be used dependably to improve performance of crops grown in nonfumigated soil.
This invention provides a signal that produces propagule germination and subsequent root colonization of mycorrhizae in a very surprising way. It has long been known that seeds store phosphorus as phytate (IP6) and that a germinating seed produces the enzyme phytase to break down the phytate into plant-useable forms to provide nutrients for the seedling. It has also been known that the breakdown of phytate (a six phosphorus molecule) by the enzyme phytase releases three moles of inorganic phosphorus (orthophosphate) and myo-inositol triphosphate (IP3). Plants need phosphorus in an inorganic form, primarily orthophosphate, to take the nutrient into the root. Plants use very little organic phosphorus as they do not possess an effective method to break down phytate. Myo-inostitol triphosphate (IP3) is known as a second messenger that can facilitate communications and/or responses between organisms. The release of myo-inositol that occurs through hydrolysis of phytate with B. amyloliquefaciens phytase has an impact on plant-microbe interactions and specifically interactions between plants and N fixing bacteria. The signal that is responsible for the germination of mycorrhizae and the subsequent colonization of the plant root by mycorrhizal fungi is unknown. In addition, the IP3 signal has not been suggested in the literature as having any link to mycorrhizal fungi response, propagule germination, or root colonization. In fact, it is well known that mycorrhizae root colonization can be achieved in low phosphorus soil conditions but it is extremely difficult to produce mycorrhizal germination and root colonization in high phosphorus soil conditions or high phosphorus rhizosphere environment. It is, therefore, also a fact that the literature teaches away from the notion that using a phytase enzyme to reduce phytate and release readily-plant-available phosphorus in the rhizosphere would result in a signal that facilitates germination and subsequent colonization of plant roots by mycorrhizal fungi.
It is likely that additional study of this invention will produce dual and perhaps multiple signal mechanisms as it is known that germination of mycorrhizae propagules can occur in the absence of the plant root; however, the propagule germination is more likely when the root is present. This suggests an unknown signal response.
The background art is characterized by U.S. Pat. Nos. 4,476,881; 4,489,161; 4,642,131; 4,668,512; 4,678,669; 4,713,342; 4,724,147; 4,748,021; 4,818,530; 4,828,600; 4,877,738; 4,915,944; 4,952,229; 5,047,239; 5,049,379; 5,071,462; 5,068,105; 5,084,272; 5,194,258; 5,238,690; 5,260,213; 5,266,316; 5,273,749; 5,300,127; 5,344,647; 5,401,655; 5,422,107; 5,455,028; 5,409,509; 5,552,138; 5,589,381; 5,614,188; 5,628,144; 5,632,987; 5,645,831; 5,665,354; 5,667,779; 5,695,982; 5,702,701; 5,753,222; 5,852,054; 5,869,042; 5,882,641; 5,882,915; 5,906,818; 5,916,029; 5,919,447; 5,922,603; 5,972,689; 5,974,734; 5,994,117; 5,998,196; 6,015,553; 6,017,525; 6,030,610; 6,033,659; 6,060,051; 6,103,228; and 7,339,091; the disclosures of which patents are incorporated by reference as if fully set forth herein.